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Causes Medium-Chain 3 bioRxiv preprint doi: https://doi.org/10.1101/2021.03.02.433634; this version posted March 3, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. Murine deficiency of peroxisomal L-bifunctional protein (EHHADH) causes medium-chain 3- hydroxydicarboxylic aciduria and perturbs hepatic cholesterol homeostasis Pablo Ranea-Robles1, Sara Violante1,2, Carmen Argmann1, Tetyana Dodatko1, Dipankar Bhattacharya3, Hongjie Chen1,4, Chunli Yu1,4, Scott L. Friedman3, Michelle Puchowicz5,6, Sander M. Houten1 1Department of Genetics and Genomic Sciences, Icahn Institute for Data Science and Genomic Technology, Icahn School of Medicine at Mount Sinai, New York, NY 10029, USA 2The Donald B. and Catherine C. Marron Cancer Metabolism Center, Memorial Sloan Kettering Cancer Center, New York, NY 10065, USA. 3Division of Liver Diseases, Department of Medicine, Icahn School of Medicine at Mount Sinai, New York, NY 10029, USA. 4Mount Sinai Genomics, Inc, Stamford, CT 06902, USA. 5Department of Nutrition, School of Medicine, Case Western Reserve University, Cleveland, OH 44106 USA. 6Department of Pediatrics, University of Tennessee Health Science Center, Memphis, TN 38163, USA. Short title: The role of peroxisomal L-bifunctional protein Address for correspondence: Sander Houten, Department of Genetics and Genomic Sciences, Icahn School of Medicine at Mount Sinai, 1425 Madison Avenue, Box 1498, New York, NY 10029, USA. Phone: +1 212 659 9222, E-mail: [email protected] 1 bioRxiv preprint doi: https://doi.org/10.1101/2021.03.02.433634; this version posted March 3, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. Abstract Peroxisomes play an essential role in the β-oxidation of dicarboxylic acids (DCAs), which are metabolites formed upon ω-oxidation of fatty acids. Genetic evidence linking transporters and enzymes to specific DCA β-oxidation steps is generally lacking. Moreover, the physiological functions of DCA metabolism remain largely unknown. In this study, we aimed to characterize the DCA β-oxidation pathway in human cells, and to evaluate the biological role of DCA metabolism using mice deficient in the peroxisomal L- bifunctional protein (Ehhadh KO mice). In vitro experiments using HEK-293 KO cell lines demonstrate that ABCD3 and ACOX1 are essential in DCA β-oxidation, whereas both the bifunctional proteins (EHHADH and HSD17B4) and the thiolases (ACAA1 and SCPx) have overlapping functions and their contribution may depend on expression level. We also show that medium-chain 3-hydroxydicarboxylic aciduria is a prominent feature of EHHADH deficiency in mice most notably upon inhibition of mitochondrial fatty acid oxidation. Using stable isotope tracing methodology, we confirmed that products of peroxisomal DCA β-oxidation can be transported to mitochondria for further metabolism. Finally, we show that, in liver, Ehhadh KO mice have increased mRNA and protein expression of cholesterol biosynthesis enzymes with decreased (in females) or similar (in males) rate of cholesterol synthesis. We conclude that EHHADH plays an essential role in the metabolism of medium-chain DCAs and postulate that peroxisomal DCA β-oxidation is a regulator of hepatic cholesterol biosynthesis. Keywords multifunctional enzyme 1; fatty acid oxidation; omega-oxidation; dodecanedioic acid; hexadecanedioic acid 2 bioRxiv preprint doi: https://doi.org/10.1101/2021.03.02.433634; this version posted March 3, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. Introduction Fatty acid β-oxidation (FAO) constitutes the primary source of energy during metabolically demanding conditions such as fasting or exercise (Houten et al. 2016). The major FAO pathway is mitochondrial, but peroxisomes also harbor FAO pathways that metabolize specific carboxylic acids such as very long-chain fatty acids, branched-chain fatty acids, bile acids and long-chain dicarboxylic acids (DCAs) (Wanders & Waterham 2006). These DCAs are products of fatty acid ω-oxidation, an alternative pathway for fatty acid metabolism initiated by microsomal cytochrome P450 enzymes of the CYP4A family (Hardwick et al. 1987; PREISS & BLOCH 1964). CYP4A enzymes catalyze the ω-hydroxylation of long-chain fatty acids that are subsequently converted into long-chain DCAs by the successive action of an alcohol and an aldehyde dehydrogenase. Peroxisomal β-oxidation of DCAs generates unique products such as adipic and succinic acid (Jin et al. 2015; Rusoff et al. 1960; Tserng & Jin 1991a), the latter being able to enter the TCA cycle. In addition, peroxisomal β-oxidation yields acetyl-CoA units, which have a variety of fates including lipogenesis, ketogenesis, or oxidation in the TCA cycle. These metabolic pathway connections suggest an important role for DCA β-oxidation in intermediary metabolism. Mitochondrial and peroxisomal FAO as well as ω-oxidation are usually induced in parallel, particularly in the liver and the kidney, under conditions of high lipid flux such as during fasting (Mortensen & Gregersen 1981; Pettersen et al. 1972). Notably, medium-chain dicarboxylic aciduria; i.e. the excretion of C6-, C8-, and C10-DCAs (adipic, suberic and sebacic acids, respectively) in the urine, is a feature shared between fasting individuals (ketosis) and patients with inherited defects in mitochondrial FAO (Fontaine et al. 1998; Gregersen et al. 1980; Karpati et al. 1975; Przyrembel et al. 1976; Röschinger et al. 2000; Vianey-Saban et al. 1998). These findings further illustrate the important role of fatty acid ω- oxidation and subsequent DCA β-oxidation under lipid metabolic stress. However, the physiological roles of DCA metabolism remain largely to be established. 3 bioRxiv preprint doi: https://doi.org/10.1101/2021.03.02.433634; this version posted March 3, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. DCA metabolism begins with their activation by a dicarboxylyl-CoA synthetase (Vamecq et al. 1985). Subsequent DCA β-oxidation is thought to occur in peroxisomes (Dirkx et al. 2007; Ferdinandusse et al. 2004; Kølvraa & Gregersen 1986; Suzuki et al. 1989). Both EHHADH (also known as peroxisomal L-bifunctional protein, LBP) and HSD17B4 (also known as D-bifunctional protein, DBP) can catalyze the second and third steps of peroxisomal FAO. We and others have demonstrated that EHHADH plays a prominent role in the oxidation of long-chain DCAs (Ding et al. 2013; Dirkx et al. 2007; Ferdinandusse et al. 2004; Houten et al. 2012; Nguyen et al. 2008). Other peroxisomal proteins that participate in DCA metabolism are the ABCD3 transporter (van Roermund et al. 2014), ACOX1 (acyl-CoA oxidase 1) (Ferdinandusse et al. 2004; Poosch & Yamazaki 1989), and the thiolases ACAA1 (3-ketoacyl-CoA thiolase) and SCPx (sterol carrier protein X) (Ferdinandusse et al. 2004). In many cases, genetic evidence that links the encoding genes to DCA β-oxidation is lacking. In summary, ω-oxidation and subsequent peroxisomal DCA β-oxidation is an alternate pathway for fatty acid metabolism that does not rely on the classic mitochondrial FAO pathway. In this study, we assessed the contribution of candidate peroxisomal proteins to DCA β-oxidation using KO HEK-293 cell lines created by CRISPR-Cas9 genome editing. We also examined the physiological role of DCA β-oxidation through the study of the Ehhadh KO mouse model. Finally, we assessed the relevance of peroxisomal DCA metabolism in mouse models with mitochondrial FAO defects. The results of our study further highlight an important role for peroxisomal DCA β-oxidation in intermediary metabolism. 4 bioRxiv preprint doi: https://doi.org/10.1101/2021.03.02.433634; this version posted March 3, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. Material and Methods Materials HEK-293 cells (ATCC® CRL-1573™) were obtained from the American Type Culture Collection (Manassas, VA, USA). Dulbecco’s modified Eagle’s medium (DMEM), Krebs–Henseleit buffer, William’s E Medium with GlutaMAX™ Supplement (WEGG), heat-inactivated fetal bovine serum (FBS), penicillin, streptomycin, and Lipofectamine 2000 were purchased from Thermo Fisher Scientific (Waltham, MA, USA). CRISPR-Cas9 plasmid [pSpCas9(BB)-2A–green fluorescent protein (GFP); PX458] (Ran et al. 2013) was obtained from Addgene (Watertown, MA, USA). The blocking buffer and the fluorescent secondary anti-rabbit and anti-mouse IgG antibodies were obtained from LI-COR 2 2 Biosciences (Lincoln, NE, USA). [ H]-water (99% enriched), the L-carnitine internal standard ( H9- 2 2 carnitine) and the acylcarnitine internal standard mix containing H9-carnitine, H3-acetylcarnitine (C2), 2 2 2 2 H3-propionylcarnitine (C3), H3-butyrylcarnitine (C4), H9-isovalerylcarnitine (C5), H3- 2 2 octanoylcarnitine (C8), H9-myristoylcarnitine (C14), and H3-palmitoylcarnitine (C16), were from Cambridge Isotope Laboratories (Tewksbury, MA, USA). Minimal essential medium, bovine serum albumin (fatty acid free), L-carnitine, lauric acid (C12:0), [U-13C]-dodecanedioic acid ([U-13C]-C12- DCA, 99 atom % 13C), dodecanedioic acid (C12-DCA), and hexadecanedioic
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